Method for early diagnosis of liver cancer

ABSTRACT

Disclosed is a method for early diagnosis of liver cancer. The method comprises the steps of:(A) providing a sample obtained from a subject; (B) assessing the expression level of four subtypes of α-mannosidase genes consisting of MAN1C1 in the sample; (C) comparing the expression level of α-mannosidase genes in the sample with a normal control; and (D) determining whether the subject having a risk of suffering liver cancer in accordance with the result of step (C); wherein while the MAN1C1 expression level of the sample is lower than that in the normal control, the subject is determined to have a risk of suffering liver cancer. Additionally, while MAN1A1, MAN1A2 and MAN1B1 expression levels in the sample are higher than those in control group, the subject is determined to suffer from liver cancer and has a risk of metastasis. In the future, MAN1C1 can be applied to early diagnosis of liver cancer and metastasis, suppression of liver metastasis, and screening agents for treating liver cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/220,055, filed Aug. 29, 2011, which claims priority under 35 U.S.C.§119(a) on Patent Application No(s). 100126558 filed in Taiwan, Republicof China, on Jul. 27, 2011, the entire contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a method for diagnosis of liver cancerand metastasis, particularly relates to a method using MAN1C1 for earlydiagnosis of liver cancer, inhibition of metastasis, and screening drugsfor treating liver cancer.

BACKGROUND OF THE INVENTION

Liver cancer is one of the most common malignant tumors in Taiwan, morethan 7,000 people died as a result of liver cancer each year. Thesymptom was not obvious in early stage; the patients feel nothing afterhaving liver cancer for long time. Until the progression of the diseaseto some degree, it will gradually produce some symptoms such as liverpain, loss of appetite, fatigue, weakness, losing weight etc. At thelater stage, patients develop jaundice, ascites, vomiting, coma andother symptoms. Patients with liver cancer often palpable huge tumor onabdominal, however this has come in late, and even metastasis to thelungs and other organs. The overall duration of liver cancer is abouttwo and half years, of which first two years are the early stage withoutsymptoms. Once the symptoms appear, the survival time is only sixmonths. Liver cancer is very difficult to diagnosis. For most of thepatients, liver transplantation is their only hope. The method for earlydiagnosis could save countless lives. Some of the current method ofdetection of tumor growth is often based on the existence of the bloodconcentrations of specific markers. For the detection of liver cancer,commonly use α-fetoprotein (AFP) in diagnosing liver cancer. AFP is anormal fetal serum protein synthesized by the liver, yolk sac, andgastrointestinal tract that shares sequence homology with albumin. It isa major component of fetal plasma, reaching a peak concentration of 3mg/ml at 12 weeks of gestation. AFP can be found in 95% primary livercancer patients' blood, it is also used as a marker for screening livercirrhosis and hepatitis. Due to AFP's low specificity, fake positiveresults are frequently occurs. It is estimated that 6 billion NTDcommercial potential exist in Taiwan's market regarding liver canerearly diagnosis, and a more gigantic potential exists in foreign market.

The process of N-glycosylation consists of a covalent linkage of aspecific oligosaccharide (Blc3Man9GlcNAc2) on a nascent protein. Oncethe oligosaccharide is transferred, several subsequent steps ofmaturation will occur along the secretory pathway. N-glycosylation isubiquitous in eukaryotes. First steps of N-glycosylation are conservedthrough eukaryotes from yeast to human, which take place in theendoplasmic reticulum. The following and last steps of maturationleading to polymannosylated glycoprotein, which occur in the Golgiapparatus, and are species specific. The function of α-mannosidase is totrim the mammose of the glycoprotein in the process of N-glycosylation.There are many types of α-mannosidase in human. Previous studiesrevealed that some specific types of mannosidase are related to theformation of cancer, supported with high expression level of mannosidasein particular cancer. Swainsonine (SW), α-mannosidase II inhibitor canefficiently decrease the tumor size in nude mice injected with leukemiacell (MDAY-D2) (Goss, 1995). Deoxymannojirimyci (DMJ), α-mannosidase Iinhibitor decreased migration ability of bladder cancer cells (T24)(Przybylo, 2005). DMJ also can induce liver cancer cell (7721) towardapoptosis (Przybylo, 2005). Based on these literatures, the presentinvention further discover the expression level of four α-mannosidasegenes in different stages of liver cancer and their correlation tomigration ability. Furthermore, early diagnosis of cancer usingα-mannosidase has not been reported previously, and we identified onetype of α-mannosidase-MAN1C1 can predict the early stage.

SUMMARY OF THE INVENTION

Though high expression level of α-mannosidase has been known to beassociated with specific cancers, and suppressing the activity ofα-mannosidase may inhibit growth, induce apoptosis even decreasemigration ability of cancer cells. However, early diagnosis of livercancer using MAN1C1 has not been reported before. Furthermore,expression levels of four α-mannosidase subtypes have never beenidentified in different liver cancer stages.

One object of the present invention is to provide a method for earlydiagnosis of liver cancer by low expression of MAN1C1.

Another object of the present invention is to provide a method fordetermining liver cancer and metastasis by high expression of MAN1A1,MAN1A2 and MAN1B1.

Yet another object of the present invention is to provide a method forinhibiting metastasis by overexpressing MAN1C1 in liver cancer cells.

Yet another object of the present invention is to provide a marker forscreening target drug for treating liver cancer.

In one embodiment, the method for early diagnosis of liver cancercomprises the steps of: (A) providing a sample obtained from a subject;(B) assessing the expression level of four subtypes of α-mannosidasegenes consisting of MAN1A1, MAN1A2, MAN1B1 and MAN1C1 in the sample; (C)comparing the expression level of α-mannosidase genes in the sample witha normal control; and (D) determining whether the subject having a riskof suffering liver cancer in accordance with the result of step (C);wherein while the MAN1C1 expression level of the sample is lower thanthat in the normal control, the subject is determined to have a risk ofsuffering liver cancer. Additionally, while MAN1A1, MAN1A2 and MAN1B1expression levels in the sample are higher than those in control group,the subject is determined to suffer from liver cancer and has a risk ofmetastasis.

Preferably, the expression levels of MAN1A1, MAN1A2, MAN1B1 and MAN1C1in the sample are at least two folds higher or lower than those in thenormal control; wherein step (D) further comprises comparing MMP9expression level in the sample with a normal control, while MAN1A1,MAN1A2 and MAN1B1 expression levels in the sample are higher than thosein control group, and the MMP9 expression level in the sample is higherthan in the normal control, the subject is determined to have a risk ofliver metastasis. The expression level of α-mannosidase (MAN1A1, MAN1A2,MAN1B1 and MAN1C1) and MMP9 mentioned above can be either RNA orprotein, and the subject is hepatitis B virus carrier, and the sample isa liver tissue obtained from the subject.

In another embodiment, the method of inhibiting metastasis in livercancer cell comprises a step of overexpressing MAN1C1 in a liver cancercell so as to inhibit liver metastasis. Preferably, overexpressingMAN1C1 can inhibit the MMP9 expression level in the liver cancer cell.

In yet another embodiment, the method of screening a drug for livercancer, comprises the steps of: (A) providing a liver cancer celltreated with a drug; (B) assessing MAN1C1 expression level of the livercancer cell; (C) determining whether the drug has a therapeutical effectaccording to the MAN1C1 expression level.

In the future, MAN1C1 can be applied to early diagnosis of liver cancerand metastasis, suppression of liver metastasis, and screening agentsfor treating liver cancer.

The embodiments of the present invention are further described throughbelow detailed examples and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the Golgi N-linked carbohydrate processing pathway.

FIG. 2A-2B demonstrate a flowchart of experiments in the presentinvention.

FIG. 3A-3E demonstrates MAN1A1, MAN1A2, MAN1B1, MAN1C and MAN1C1expression levels using the samples of human liver cancer patients carryhepatitis B virus.

FIG. 4 demonstrates the Q-PCR result of endogenous MAN1A1, MAN1A2,MAN1B1, and MAN1C1 expression levels in different cell lines.

FIG. 5A-5E demonstrate the cell migration assay results afteroverexpression of MAN1A1 and MAN1C1.

FIG. 6A-6E demonstrate the cell migration assay results after shRNAknockdown of MAN1A1, MAN1A2 and MAN1B1.

FIG. 7A-7C demonstrate the cell migration assay results after stableoverexpression of MAN1C1 in Hep3B (MAN1C1/Hep3B).

FIG. 8 demonstrate the in vivo cell migration assay results of 293T,PLC5 and Hep3B cells by xenotransplantation into zebrafish.

FIG. 9 demonstrate the cell migration assay results ofMAN1C1-overexpressed Hep3B cells (MAN1C1/Hep3B) by in vivoxenotransplantation into zebrafish.

FIG. 10A-10B demonstrate the MTT assay result.

FIG. 11A-11B demonstrate MMP9 expression level in MAN1C1-overexpressedcells.

DETAILED DESCRIPTION

A method for early diagnosis of liver cancer and prediction ofmetastasis is described with reference to the preferred embodimentsbelow, it is apparent to those skilled in the art that a variety ofmodifications and changes may be made without departing from the scopeof the present invention which is intended to be defined by the appendedclaims.

FIG. 1 demonstrates the Golgi N-linked carbohydrate processing pathway.As shown in FIG. 1, α-1, 2 mannosidase I plays a role for trimmingcarbohydrate branches in carbohydrate processing. The inhibitors such asDMJ and SW can inhibit oligosaccharide chain trimming. Oligosaccharidechain synthesis and processing includes a series of glycosidehydrolases, such as glucosidase (i.e. glucosidase II) and mannosidase(i.e. mannosidase I and mannosidase II). Mannosidase I and mannosidaseII mainly present in Golgi apparatus, these enzymes can be inhibited andterminate the Golgi N-linked carbohydrate processing, so as to producehigh-mannose and complex-type N-linked glycans. α-1, 2 mannosidase Iinhibitor DMJ and α-mannosidase II inhibitor SW can influence glycanepitope expression and further inhibit tumor cell migration, invasionand growth in vitro, which shows a potential ability of inhibitingcancer metastasis.

FIG. 2A-2B demonstrate a flowchart of experiments in the presentinvention. As shown in FIG. 2A, the quantitative PCR was performed toassess endogenous α-1, 2 mannosidase mRNA in mice and human. As shown inFIG. 2B, MAN1A1, MAN1A2, MAN1B1 and MAN1C1 cloning was performed.Overexpression and shRNA knockdown of α-1, 2 mannosidase were performedin the in vitro cell migration assay. Also a MAN1C1 stable transfectedcell line was established and injected into zebrafish embryo, and an invivo cell migration assay was then performed. Simultaneously, Q-PCRexperiment was performed to assess if MMP9 mRNA level has changed due toα-1, 2 mannosidase overexpression.

FIG. 3 demonstrates MAN1A1, MAN1A2, MAN1B1, MAN1C and MAN1C1 expressionlevels in liver tissue obtained from hepatitis B virus positive livercancer patients. As shown in FIG. 3A-3E, MANA1, MANA2, MANB1, MAN1C andMAN1C1 mRNA level of liver cancer patients were assessed by Q-PCR,wherein MANIC was part of MAN1C1. Those patients were separated intothree stages: stages I, II and III, the latter stage represents the moreaggressive cancerous condition. The cancerous liver tissue was used asexperiment group, and the non-cancerous liver tissue obtained from thesame patient was used as a control group. In cancerous tissue, two-foldexpression higher than control group was defined as overexpression, andtwo-fold expression lower than control group was defined as decreasedexpression. As shown in FIG. 3A-3E, MAN1A1, MAN1A2 and MAN1B1 wereelevated aggressive cancerous condition, particularly two folds thannormal liver tissue. However, MAN1C1 expression in the early stage livercancer patient was lower than that in normal liver tissue with 2 folds.

FIG. 4 demonstrates the Q-PCR result including: (A) endogenous MAN1A1,MAN1A2 and MAN1B1 RNA levels; performing in vitro cell migration assayin different cell lines (B) PLC5 and (C) Hep3B, to determined which cellline for further overexpression and knockdown experiments (D) endogenousMAN1C1 RNA levels in different cell lines. A serial dilution (10⁻³˜10⁻⁹)was performed by using green fluorescent protein (GFP) DNA with knownmolecule numbers to be a standard curve. By using the standard curve, Ctvalue was calculated to molecule numbers by interpolation, such thatendogenous MAN1A1, MAN1A2, MAN1B1 and MAN1C1 mRNA numbers in PLC5, Hep3Band HepG2 cells were obtained. 10⁵ cells were added in 3000 of serumfree DMEM and incubated on a transwell culture dish which had a membranewith 6.4 mm diameter and 8.0 μm pore size. 500 μl medium (10% FBS inDMEM) was added into lower chamber, and the dish was incubated at 37° C.for 48 hours, and the cells in the lower chamber was stained with 1×DAPI and calculated. The result was shown in figure, endogenous α-1, 2mannosidase mRNA levels were varied in different cells, and themigration ability of Hep3B was greater than PLC5. According toendogenous mRNA level and cell migration ability (E), PLC5 cell line wasdetermined to be used in MAN1A1 overexpression experiment, Hep3B wasdetermined to be used in MAN1A1, MAN1A2 and MAN1B1 knockdownexperiments, and MAN1C1 overexpression was performed in Hep3B.

FIG. 5A-5E demonstrate the cell migration assay results afteroverexpression of MAN1A1 and MAN1C1 for two days. As shown in FIG. 5A,Q-PCR was performed to quantify MAN1A1 and MAN1C1 mRNA expression level,and untransfected cells (mock) were used as control. Cell migrationassay results were shown in FIGS. 5B, 5C, 5D and 5E, and the method usedwas similar to FIG. 4B and 4C. Results shown in FIG. 5A demonstratedthat MAN1A1 and MAN1C1 mRNA were successfully elevated to 11.73 and160278 folds as compared with Hep3B. Furthermore, MAN1A1 overexpressionadvanced the ability of cell migration to 2.41 folds, and MAN1C1overexpression reduced the ability of cell migration to 0.48 folds. FIG.5B and 5C are photos which demonstrate cell migration assay results ofPLC5 and MAN1A1 overexpression in PLC5. FIG. 5D and 5E are photos whichdemonstrate cell migration assay results of Hep3B and MAN1C1overexpression in Hep3B.

FIG. 6A-6E demonstrate the cell migration assay results after shRNAknockdown of MAN1A1, MAN1A2 and MAN1B1 for two days. As shown in FIG.5A, Q-PCR was performed to quantify MAN1A1, MAN1A2 and MAN1B1 mRNAexpression level, and untransfected cells (mock) were used as control.Cell migration assay results were shown in FIGS. 6B, 6C, 6D and 6E, andthe method used was similar to FIG. 4B and 4C. Results shown in FIG. 6Ademonstrated that MAN1A1, MAN1A2 and MAN1B1 mRNA after knockdown withshRNA were successfully reduced to 55%, 62% and 64% as compared withuntransfected Hep3B cells. Furthermore, MAN1A1, MAN1A2 and MAN1B1knockdown reduced the ability of cell migration to 31%, 38% and 45% ascompared with untransfected Hep3B cells. FIG. 6B-6E are photos whichdemonstrate cell migration assay results of Hep3B before (B) and aftershRNA knockdown of MAN1A1(C), MAN1A2(D) and MAN1B1(E).

FIG. 7A-7C demonstrate the cell migration assay results after stableoverexpression of MANIC1 in Hep3B (MAN1C1/Hep3B). Stable overexpressedHep3B was used as experiment group, and original Hep3B was used ascontrol group. Methods used in FIG. 7A: Q-PCR was performed to quantifyMAN1C1 mRNA expression level, and untransfected cells (mock) were usedas control. The cell migration assay performed in FIGS. 7B and 7C weresimilar to FIGS. 4B and 4C, except the analyzing time was after 24 hr.As shown in figures, cell migration ability of cells with stableoverexpression of MAN1C1 was reduced to 16% as compared with originalHep3B. FIGS. 7B and 7C are photos which demonstrate cell migration assayresults of Hep3B and stable overexpression of MAN1C1 in Hep3B.

FIG. 8 demonstrate in vivo cell migration assay results of 293T, PLC5and Hep3B cells by xenotransplantation to zebrafish embryos. 293T is atransformed human kidney cell line. PCL5 and Hep3B are human hepatomacell lines. Establishment of the xenotransplantation zebrafish animalmodel in FIG. 8: using DiI to label the suspension of cells andincubated in PBS before injection, and the labeled cells weremicroinjected into 2 days old zebrafish embryos. Each injection amountwas 4.6 nl and contained 400 cells, and the injection position was therear of yolk. The injected cells were monitored for 3 days afterinjection. Previous in vitro experiment results found that migrationability of Hep3B was greater than PLC5, and the identical conclusion wasonce identified by in vivo experiment, and the 293T were non-cancerouscontrol. According to statistic analysis results, cell migration wasobserved in 3.33% of zebrafish after injection of 293T cells. Three daysafter injection, cell migration was observed in 62% of zebrafish afterinjection of Hep3B cells. These results were consistent with theprevious in vitro experiments. After 3 days of microinjection, 293T andPLC5 cells were still retained in yolk cavity. The position of yolkcavity where Hep3B cells injected. The cells were found to migrate totail after 1 day of injection.

FIG. 9 demonstrate the cell migration assay results ofMAN1C1-overexpressed Hep3B cells (MAN1C1/Hep3B) by in vivoxenotransplantation. Stable overexpression of MAN1C1 in Hep3B was usedas experiment group, and Hep3B was used as control. The cells ofexperiment group and control were microinjected into zebrafish yolk.Each injection contains 400 labeled cells, and these cells were observedand recorded at 1^(St) and 3^(rd) day. The method used was similar toFIG. 8, however experiment group was stable overexpressed MAN1C1 Hep3B,and the control contained large amount of expression vectors. Accordingto statistic analysis results, cell migration ability was 18.6% inexperiment group, and cell migration ability was 36.2% in control group.According to the results, three days after injection, cell migrationability of stable overexpressed MAN1C1 in Hep3B was two folds thanoriginal Hep3B. The cells were found to migrate to tail in control groupafter 1 day of injection, this result was also found after 3 days ofinjection. 80% of MAN1C1/Hep3B cells were observed that Hep3B cells wereretained in yolk cavity after 3 days of injection.

FIG. 10 demonstrates MTT assay result. X-axis represents time (hour),and Y-axis represents proliferation fold. Experiment method: 1. 6,000cells were seeded on a 96-well culture dish. 2. 200 μl of mixed medium(MTT:DMEM=1:9) was added into the dish every 24 hr, and cells wereincubated for 3 hr at 37° C. 3. 100 μl of DMSO was used to break thecells, and absorbance value (OD_(570nm)) of each sample was measured.Experiment group: overexpression of MAN1A1 in Hep3B (A) or stableoverexpression of MAN1C1 in Hep3B (B). Control group: nontransfectedHep3B cells. As shown in MTT assay results of FIG. 10A, overexpressionof MAN1A1 promotes Hep3B cells growing faster than nontransfected cells.As shown in FIG. 10B, the growth curves of experiment group(overexpression of MAN1C1) and control has no difference.

FIG. 11A-11B demonstrate MMP9 expression level in MAN1A1 orMAN1C1-overexpressed cells. As shown in FIG. 11A, Q-PCR was used toassess MMP9 expression level, untransfected cells were used as controland expression more than 2 folds was defined as overexpression. Theresults suggest overexpression of MAN1A1 increased MMP9 expression to2.3 folds than control, and overexpression of MAN1C1 suppressed MMP9expression to 0.33 folds. As shown in FIG. 11B, RT-PCR was performed toamplify MAN1C1 and MMP9 cDNA, and 18s RNA was used as control. MMP9expression in stable overexpression MAN1C1 Hep3B cellsd was reduced incomparison with control group.

As results disclosed in the present invention, three genes: MAN1A1,MAN1A2 and MAN1B1 were overpressed in liver cancer when compare to thenormal counterpart. However, the expression of MAN1C1 was down-regulatedin HCC patients when compare to normal liver tissues. Moreover, 94% ofHBV carrier HCC patients exhibit over two-fold decreased MAN1C1expression as early as stage I. This result indicated that thedecreasing expression of MAN1C1 might be a potential biomarker for earlydiagnosis for HCC. Those expression patterns implied MAN1A1, MAN1A2 andMAN1B1 probably are potential oncogenes, and the MAN1C1 might functionsas tumor suppressor. In order to study the role of four α-mannosidasegenes during hepatocarcinogenesis, we first cloned the genes for MAN1A1,MAN1A2, MAN1B1 and MAN1C1, and used cell line to investigate theproliferation, migration and other genes' expression afterover-expressed or knockdown those genes. It was found thatoverexpression of MAN1A1 into PLC5 cells can enhance the migrationability, and knockdown of MAN1A1, MAN1A2 and MAN1B1 can decrease themigration ability in Hep3B cells. On the other hand, overexpressionMAN1C1 in Hep3B cell decreased migration ability by in vitro transwellassay. To further determine how α-1, 2 mannosidase I influencedmigration ability, hepatic cell lines with stable overexpression of α-1,2 mannosidase I were thus established. Zebrafish embryo was used toperform in vivo xenotransplantation to observe hepatic cancerous cellsmigration in vivo, and found that migration ability of MAN1C1/Hep3Bstable cell line was reduced in zebrafish embryo. To further study therelationship between α-mannosidase genes and cell migration, we focus onmatrix metalloproteinases (MMPs) which are proteases to promoted cancercells growth, migration, invasion and metastasis (Egeblad and Werb,2002). According to Q-PCR results, it was suggested that overexpressionof MAN1A1 increased MMP9 mRNA expression level, and overexpression ofMAN1C1 decreased MMP9 mRNA expression level. Due to MMPs are capable ofdegrading all kinds of extracellular matrix proteins, decreased MMP9expression means that cell migration and invasion ability is inhibited.According to disclosure of the present invention, it is demonstratedthat early reduction of MAN1C1 overexpression in liver cancer patientshas potential to be a molecular marker for screening early liver cancer.As proved in cell migration assay, no matter in vivo or in vitroexperiment results suggest that MAN1C1 is capable of inhibiting cellmigration ability of hepatic cancerous cells.

In conclusion, MAN1C1 has potential to be a tumor suppressor gene andapply to early diagnosis for liver cancer. In one embodiment, the methodfor early diagnosis of liver cancer comprises the steps of:(A) providinga sample obtained from a subject; (B) assessing the expression level offour subtypes of α-mannosidase genes consisting of MAN1A1, MAN1A2,MAN1B1 and MAN1C1 in the sample; (C) comparing the expression level ofα-mannosidase genes in the sample with a normal control; and (D)determining whether the subject having a risk of suffering liver cancerin accordance with the result of step (C); wherein while the MAN1C1expression level of the sample is lower than that in the normal control,the subject is determined to have a risk of suffering liver cancer.Additionally, while MAN1A1, MAN1A2 and MAN1B1 expression levels in thesample are higher than those in control group, the subject is determinedto suffer from liver cancer and has a risk of metastasis.

Preferably, the expression levels of MAN1A1, MAN1A2, MAN1B1 and MAN1C1in the sample are at least two folds higher or lower than those in thenormal control; wherein step (D) further comprises comparing MMP9expression level in the sample with a normal control, while MAN1A1,MAN1A2 and MAN1B1 expression levels in the sample are higher than thosein control group, and the MMP9 expression level in the sample is higherthan in the normal control, the subject is determined to have a risk ofliver metastasis. The expression level of α-mannosidase (MAN1A1, MAN1A2,MAN1B1 and MAN1C1) and MMP9 mentioned above can be either RNA orprotein, and the subject is hepatitis B carrier, and the sample is aliver tissue obtained from the subject.

In another embodiment, the method of inhibiting metastasis in livercancer cell comprises a step of overexpressing MAN1C1 in a liver cancercell so as to inhibit liver metastasis. Preferably, overexpressingMAN1C1 can inhibit the MMP9 expression level in the liver cancer cell.

In yet another embodiment, the method of screening a drug for livercancer, comprises the steps of: (A) providing a liver cancer celltreated with a drug; (B) assessing MAN1C1 expression level of the livercancer cell; (C) determining whether the drug has a therapeutical effectaccording to the MAN1C1 expression level.

Although the present invention is described with reference to thepreferred embodiments thereof, it is apparent to those skilled in theart that a variety of modifications and changes may be made withoutdeparting from the scope of the present invention which is intended tobe defined by the appended claims.

What is claimed is:
 1. A method for early diagnosis of liver cancer,comprising the steps of: (A) providing a sample obtained from a subject;(B) assessing MAN1C1 expression levels in the sample by detecting MAN1C1RNA or protein expression levels in the sample; (C) comparing the MAN1C1expression levels in the sample with MAN1C1 expression levels in anormal control; and (D) determining whether the subject having a risk ofsuffering liver cancer in accordance with the result of step (C);wherein while the MAN1C1 expression levels of the sample is lower thanthat in the normal control, the subject is determined to have a risk ofsuffering liver cancer; wherein the sample and the normal control areliver biopsies.
 2. The method as claimed in claim 1, wherein the MAN1C1expression levels in the sample are at least two folds lower than theMAN1C1 expression levels in the normal control.
 3. The method as claimedin claim 1, wherein the subject is hepatitis B virus carrier.
 4. Amethod of inhibiting metastasis of liver cancer, comprising a step ofoverexpressing MAN1C1 by increasing α-mannosidase IC expression levelsin a liver cancer cell.
 5. The method as claimed in claim 4, whereinoverexpressing MAN1C1 can inhibit the MMP9 expression levels.
 6. Amethod of screening a drug for liver cancer, comprising the steps of:(A) providing a liver cancer cell treated with a drug; (B) assessingMAN1C1 expression levels in the liver cancer cell; (C) determiningwhether the drug has a therapeutical effect according to the MAN1C1expression levels.
 7. The method as claimed in claim 6, furthercomprising assessing MMP9 expression levels in the liver cancer cell,and determining whether the drug has a therapeutical effect according tothe MMP9 expression levels.